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Neuropharmacology 62 (2012) 1708e1716
Contents lists available
Neuropharmacology
journal homepage: www.elsevier .com/locate/neuropharm
The noradrenaline transporter as site of action for the
anti-Parkinsondrug amantadine
Christian Sommerauer a, Patrick Rebernik a, Harald Reither a,
Christian Nanoff b, Christian Pifl a,*aCenter for Brain Research,
Medical University of Vienna, Spitalgasse 4, A-1090 Vienna,
AustriabCenter for Physiology and Pharmacology, Institute of
Pharmacology, Medical University of Vienna, Wahringerstrasse 13a,
A-1090 Vienna, Austria
a r t i c l e i n f o
Article history:Received 5 September 2011Received in revised
form17 November 2011Accepted 28 November 2011
Keywords:AmantadineNoradrenaline transporterCarrier-mediated
releaseTransport-related currentsNMDA-receptorParkinson’s
disease
* Corresponding author. Tel.: þ43 1 40160 34080; fE-mail
address: [email protected] (C
0028-3908/$ e see front matter � 2011 Elsevier
Ltd.doi:10.1016/j.neuropharm.2011.11.017
a b s t r a c t
Amantadine is an established antiparkinsonian drug with a still
unclear molecular site of action. In vivostudies on rodents, in
vitro studies on tissue of rodents as well as binding studies on
post mortem humantissue implicate monoamine transporters and NMDA
receptors. In order to re-examine its action athuman variants of
these proteins on intact cells we established cells stably
expressing the human NR1/2ANMDA-receptor, noradrenaline transporter
(NAT) or dopamine transporter (DAT) and tested the activityof
amantadine in patch-clamp, uptake, release, and cytotoxicity
experiments. Amantadine was lesspotent in blockade of NMDA-induced
inward currents than in blockade of noradrenaline uptake and
ininduction of inward currents in NAT expressing cells. It was 30
times more potent in blocking uptake inNAT- than in DAT cells.
Amantadine induced NAT-mediated release at concentrations of 10e100
mM insuperfusion experiments and blocked NAT-mediated cytotoxicity
of the parkinsonism inducing neuro-toxin
1-methyl-4-phenyl-pyridinium (MPPþ) at concentrations of 30e300 mM,
whereas 300e1000 mMamantadine was necessary to block NMDA-receptor
mediated cytotoxicity. Similar to amphetamine,amantadine was
inactive at a2A-adrenergic receptors and induced reverse
noradrenaline transport byNAT albeit with smaller effect size.
Thus, amantadine acted as “amphetamine-like releaser” with
selec-tivity for the noradrenergic system. These findings and
differences with memantine, which had beenreported as less
efficient antiparkinsonian drug than amantadine but in our hands
was significantly morepotent at the NMDA-receptor, suggest
contributions from a noradrenergic mechanism in the
anti-parkinsonian action of amantadine.
� 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Since a case report on improvements of parkinsonian
symptomswhile receiving amantadine for influenza prophylaxis and
subse-quent controlled trials reporting efficacy in the treatment
ofParkinson’s disease (Parkes et al., 1970; Schwab et al.,
1969)amantadine is an established antiparkinsonian agent. It is
espe-cially useful as an adjuvant to levodopa where it
markedlyimproved motor response complications (Rajput et al.,
1998;Sawada et al., 2010; Thomas et al., 2004; Verhagen et al.,
1998) andas a drug which is available for intravenous
administration (Adleret al., 1997; Ruzicka et al., 2000). Several
modes of action havebeen proposed from in vitro experiments.
Amantadine was shownto inhibit neuronal uptake of dopamine and
noradrenaline into ratbrain homogenates (Fletcher and Redfern,
1970), slices (Heikkila
ax: þ43 1 40160 934053.. Pifl).
All rights reserved.
and Cohen, 1972) and synaptosomes (Herblin, 1972; Thornburgand
Moore, 1973). It weakly released dopamine and noradrena-line from
nerve endings isolated from rat brain (Haacke et al., 1977)and
augmented the release of dopamine at high doses in thestriatum of
rats (Papeschi, 1974; Scatton et al., 1970). An actionmediated by
post-synaptic dopamine receptors appeared unlikelysince the IC50 in
displacing the dopamine receptor ligand
[3H]N-n-propylnorapomporphine in striatal membranes was reported
tobe about 1 mM (Dunn et al., 1986). More recently, an
inhibitoryaction of amantadine at NMDA receptors has been shown;
itcompeted with [3H]MK-801 binding in membrane homogenates
ofpost-mortem human frontal cortex (Kornhuber et al.,
1991),antagonized inward current responses to NMDA in freshly
disso-ciated rat hippocampal and striatal neurons (Parsons et al.,
1996),inhibited the NMDA-evoked [3H]ACh release in slices of the
rabbitcaudate nucleus (Lupp et al., 1992), reduced NMDA
receptor-mediated neurotoxicity in cultures of rat retinal ganglion
cellneurons (Chen et al., 1992) and rat neuron-enriched
cerebrocorticalcultures (Lustig et al., 1992). Thus, experimental
evidence to date
mailto:[email protected]/science/journal/00283908http://www.elsevier.com/locate/neuropharmhttp://dx.doi.org/10.1016/j.neuropharm.2011.11.017http://dx.doi.org/10.1016/j.neuropharm.2011.11.017http://dx.doi.org/10.1016/j.neuropharm.2011.11.017
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C. Sommerauer et al. / Neuropharmacology 62 (2012) 1708e1716
1709
reflects a summary of effects obtained in various species
includingbinding data from homogenized post-mortem brain.
In Parkinson’s disease, in addition to dopamine neurons,
brain-stem noradrenergic nuclei show signs of degeneration, and
norad-renergic drugs may modify parkinsonian symptoms. Besides
thebeneficial effects of a-2 adrenoreceptor antagonists in
levodopa-induced dyskinesia (Bezard et al., 1999; Rascol et al.,
2001; Savolaet al., 2003) and the improvement of freezing gait by
the syntheticnoradrenaline precursor
L-threo-3,4-dihydroxyphenylserine(Narabayashi et al., 1987; Tohgi
et al., 1993), noradrenaline uptakeinhibitors were used in
Parkinson’s disease, not only in treatment ofdepression, but also
of motor function (Jankovic, 2009; Laitinen,1969; Marsh et al.,
2009). On the other hand, amantadine dis-played antidepressant
action in various studies (for review (Huberet al., 1999)).
The above findings motivated us to re-examine the pharma-cology
of amantadine at the noradrenaline transporter (NAT),dopamine
transporter (DAT) and the NMDA-receptor by expressingthe human
recombinant proteins in cell culture. The pharmacologyof amantadine
at the human transporters was not studied before,and studying the
human proteins seemed advisable consideringdifferences in substrate
and inhibitor affinities between rodent andhuman versions reported
previously (Giros et al., 1992; Paczkowskiet al., 1999). In cAMP
accumulation, monoamine uptake andrelease, patch-clamp and
cytotoxicity experiments only effects onthe transporters and
receptors in living cells were investigated.
2. Methods
2.1. Cell culture and molecular biology
Human embryonic kidney 293 cells were grown in minimum essential
mediumwith Earle’s salts 1-glutamine, 10% heat-inactivated fetal
bovine serum and 50 mg/lgentamicin on 60 or 100 mm tissue culture
dishes (Falcon) at 37 �C and 5% CO2/95%air. The conditions for cell
culture and transfection of cells stably expressing theporcine
a2A-adrenoceptor fused at the C-terminus to the amino terminus of
wildtype Gaie1 were described previously (Kudlacek et al., 2002).
For stable expression ofhuman DAT and NAT in HEK cells the
expression vector pRc/CMV was used asdescribed previously (Pifl et
al., 2004b). For patch-clamp experiments and for allexperiments on
NMDA-receptor expressing cells the tetracycline-regulatedexpression
system called T-REx� was used which allowed expression of
theproteins in an inducible manner (Invitrogen GmbH, Lofer,
Austria). T-REx� cellsstably expressing the tetracycline repressor
proteinwere stably transfected with therespective transporter
cDNAusing the expression plasmid pcDNA4/TO and a calciumphosphate
method as described previously (Pifl et al., 1996). Cell clones
expressingthe transporter were selected with 0.3 g/l zeocin in the
presence of 5 mg/l blasti-cidin. For expression of the human NR1/2A
NMDA receptor the cDNA of the hNR1subunit (received in bluescript
from Dr. Shigetada Nakanishi, Osaka) was subclonedinto pcDNA4/TO.
Two million cells 293/T-REx� cells were plated into 100-mmdiameter
cell culture dishes one day before transfection. At the day of
trans-fection, the medium was first changed and six to 7 h later, 1
mg of NR1/pCDNA4/TOand 5 mg hNR2A/pcDNAI (received from Dr. Antonio
Ferrer-Montiel, Elche) in 450 mlof H2O were mixed with 50 ml of
2.5M CaCl2 and the further transfection procedurewas as described
(Pifl et al., 1996). One day after transfection, plates were split
1:4 to1:8, and on the next day selection of cells was started by
adding zeocin at 0.3 g/l andblasticidin at 5 mg/l. After two weeks,
single clones were transferred with Gilsonpipette tips into 48 well
plates containingmedium and selecting antibiotics. Six dayslater
each of the cell clones was split after trypsinisation into of a
24-well anda 48-well plate, one well each. On the next day
tetracycline was added (0.1 mg/l) forinduction of the NMDA receptor
in the 48-well plate. Cell clones dying in the 48-wellplate after
adding tetracycline (0.1 mg/l) for induction of the NMDA receptor
werefurther grown up from the corresponding 24-well plate, and
receptor expressionwas verified by cell death following induction
with tetracycline and its preventionwith 200 mM memantine.
2.2. Determination of cAMP formation
Receptor-dependent inhibition of cAMP formation in stable HEK293
cells wasassessed as described previously (Bofill-Cardona et al.,
2000). Cells were seeded inpoly-D-lysine-coated 6-well plates
(2.5�105 cells/well) and 1 day later labeled with2,8-[3H]adenine (1
mCi/well) overnight, incubated in 1 ml PBS containing 100 mM ofthe
cAMP-specific phosphodiesterase-IV inhibitor Ro-201724, stimulated
withforskolin (25 mM), and receptor-dependent inhibition at RT was
determined after
a 25 min incubation with a2 adrenoceptor agonist UK 14,304 at
the indicatedconcentrations in the absence or presence of 0.1 mM
yohimbine or of 10e1000 mMamantadine. After termination by cell
lysis, [3H]cAMP was isolated by sequentialchromatography on Dowex
AG 50W-X4 and neutral alumina.
2.3. Patch clamp experiments
About 6e9� 104 cells were split into poly-D-lysine-coated 35mm
tissue culturedishes. Patch-clamp recordings were performed 3e4
days later, 18 h (NMDA-receptor) or 2e3 days (transporters) after
induction of proteins by adding tetracy-cline. The bath solution
for experiments on NMDA-receptor expressing cells was asreported by
(Monyer et al., 1992) and consisted of (mmol/l): 150 NaCl; 5.4 KCl;
1.8CaCl2; 5 HEPES; pH 7.2, NaOH. The final osmolarity was 275
mOsm/l. Patch pipetteswere filled with (mmol/l): 140 CsCl; 1 MgCl2;
10 EGTA; 10 HEPES; pH 7.2, CsOH, withan osmolarity of 303 mOsm/l.
The bath solution for experiments on NAT or DATexpressing cells
consisted of (mmol/l): 4 TriseHCl; 6.25
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); 120
NaCl; 5KCl; 1.2 CaCl2; 1.2 MgSO4; 130NaCl; 34 D-glucose and 0.5
ascorbic acid; pH 7.2. The final osmolarity was300mOsm/l. Patch
pipettes were filled with (mmol/l): 130 KCl; 0.1 CaCl2; 2 MgCl2;1.1
EGTA; 9 HEPES; 0.65 TRIS; pH 7.2, with an osmolarity of 270 mOsm/l.
Patchelectrodes were pulled from borosilicate glass capillaries
(GB150F-8P, ScienceProducts, Hofhem Germany) with a programmable
Brown-Flaming micropipettepuller (P-97; Sutter Instruments Co.,
USA)were heat-polished to a final tip resistanceof 3e6MU.
Recordingswere performed in thewhole-cell configuration of the
patch-clamp technique using an Axopatch 200B patch clamp amplifier
and the pClampdata acquisition system (Axon Instruments, Foster
City, CA, U.S.A.) at ambienttemperature (25 � 2 �C) and clamping
the cells to the holding potential of �60to �80 mV. Test drugs were
applied with a PTR-2000/DAD-12 drug applicationdevice (ALA
Scientific Instruments Inc., Westbury, NY), which allows a
completeexchange of solutions surrounding the cells under
investigationwithin
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C. Sommerauer et al. / Neuropharmacology 62 (2012)
1708e17161710
a quadruplicate of wells was determined in at least 4
independent experiments.Statistical significance was determined by
paired Student’s t-test followed byBonferroni correction comparing
the effect of amantadine with the effect of vehicle-treated cells
induced by tetracycline and, in case of NAT and DAT cells,
treatedby MPPþ.
3. Results
3.1. Amantadine had no affinity for a2 adrenoceptors
Amantadine in concentrations up to 1 mM did not modify
theconcentration-dependent inhibition of forskolin-stimulated
cAMPaccumulation by the a2 adrenoceptor agonist UK 14,304 in
HEK293cells stably expressing the porcine a2A-adrenoceptor fused at
theC-terminus to the amino terminus of wild type Gai-1, whereas0.1
mM of the prototypical a2 adrenoceptor antagonist yohimbineshifted
the concentration-response curve of UK 14,304 more than100-fold
(Fig. 1). In addition to this lack of antagonistic action at thea2
adrenoceptor, amantadine (0.1e1 mM) did not change theaccumulation
of cAMP in the absence of UK 14,304, which ruled outany agonistic
activity at this receptor as well (data not shown).
Fig. 1. Concentration-dependence of the a2 adrenoceptor agonist
UK14304 effect onforskolin-stimulated cAMP accumulation. HEK293
cells stably expressing a2A-adrenoceptor expressing were labeled
with 2,8-[3H]adenine and exposed to UK14304at the concentration
indicated in the absence (open circles) or (A) presence
ofamantadine (10 mM, triangles; 100 mM, closed circles), (B)
amantadine (1 mM, trian-gles) or yohimbine (0.1 mM, closed
circles). After a 25 min incubation [3H]cAMP wasisolated as
described under Methods. Symbols represent mean values � SEM of
3independent experiments, each in duplicates.
3.2. Amantadine blocked NMDA-induced currents at
concentrationsbeyond 30 mM
NMDA at the concentration of 100 mM induced an inwardcurrent in
HEK293 cells expressing the human NR1/NR2A receptor.Amantadine
blocked this inward current at a concentration of30 mM by 8.5 �
1.4% (n ¼ 13), at 100 mM by 32 � 7% (n ¼ 15; p < 0.5vs. 30 mM)
and at 1 mM by 72 � 8% (n ¼ 12; p < 0.5 vs. 100 mM)(Fig. 2A, B,
C). By contrast, ketamine blocked the NMDA-inducedinward current at
the concentration of 3 mM by 28 � 3% (n ¼ 6),at 10 mM by 68 � 4% (n
¼ 7; p < 0.5 vs. 3 mM) and at 30 mM by83 � 7% (n ¼ 9) (Fig. 2D,
E, F). An estimate of the potency asdescribed in Methods gave an
IC50 of 197 � 93 mM (n ¼ 5) foramantadine and 5.6 � 0.2 mM (n ¼ 3)
for ketamine.
3.3. Amantadine blocked the NAT more potently than the DAT
Amantadine blocked noradrenaline uptake by HEK293
cellsexpressing the human NAT in a concentration-dependent
manner;the IC50 of blockade was 41 � 4 mM (Fig. 3). Thirty times
higherconcentrations of amantadine were necessary to block
dopamineuptake by HEK293 cells expressing the human DAT (IC501.22 �
0.08 mM). To validate the uptake assays standard inhibitorswere
investigated under our experimental conditions. For desi-pramine
the IC50 values were 0.005 � 0.002 mM (n ¼ 3) and14.0 � 1.2 mM (n ¼
3), for cocaine 1.13 � 0.27 mM (n ¼ 4) and0.89� 0.08 mM (n¼ 8) and
for amphetamine 0.27� 0.02 mM (n¼ 4)and 0.87 � 0.16 mM (n ¼ 7) at
NAT and DAT expressing cells,respectively.
3.4. Amantadine induced inward currents in NAT expressing
cells
Blockade of monoamine uptake can be induced by pure
uptakeinhibition or by an amphetamine-like releasing effect. In
order toelucidate the type of interaction of amantadine with the
NAT, weinvestigated the action of amantadine on currents in
NATexpressing cells by patch-clamp experiments in the
whole-cellconfiguration. In these cells, 10 mM noradrenaline
induced aninward current, which was blocked by the presence of 30
mMcocaine (Fig. 4A, B). Amantadine, at the concentration of 10
mM,induced an inward current which was about 20% of that induced
by30 mMnoradrenaline (Fig. 4B). Themagnitude of inward current
didnot increase when amantadine was superfused at 100 mM (Fig.
4C)and the inward current induced by 10 mM noradrenaline
wasattenuated in the presence of 30 mM amantadine (Fig.
4D).Noradrenaline and dopamine also induced inward currents in
DATexpressing cells (Fig. 5), a carrier-mediated effect because it
wasblocked by 30 mM cocaine (shown for 10 mM NA in Fig.
5A).Amantadine, in concentrations up to 30 mM, was not able to
induceinward currents in DAT expressing cells and weakly inhibited
theeffect of 10 mM noradrenaline (Fig. 5B, C).
3.5. Amantadine induced NAT-mediated release
Since the patch-clamp experiments suggested an amphetamine-like
activity of amantadine, we tested the drug in
superfusionexperiments on HEK293 cells expressing the human NAT and
pre-loadedwith themetabolically inert transporter substrate
[3H]MPPþ.Amantadine induced tritium efflux in a
concentration-dependentmanner, with a maximum effect at 100 mM
which was about 40%of the release induced by 10 mM amphetamine
(Fig. 6A). The effectsof both, amphetamine and amantadine, were
carrier-mediated,because the release was blocked by 10 mM of the
transporterblocker mazindol (Fig. 6B, C).
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Fig. 2. Effect of amantadine or ketamine on inward currents
induced by NMDA in HEK293 cells stably expressing the NR1/2A
receptor. Cells were voltage-clamped at a holdingpotential of �60
mV and superfused for 30 s with 100 mM NMDA (upper bar) in the
absence or presence (lower bar) of amantadine (30 mM, A; 100 mM, B;
1000 mM, C) or ketamine(3 mM, D; 10 mM, E; 30 mM, F). Light/dark
grey in bars indicates change to different superfusion syringes as
a control for potential artifacts of switches. Shown are mean
values ofcurrent traces � SEM (grey area) of 12e15 (AeC) or 6e9
(DeF) cells.
C. Sommerauer et al. / Neuropharmacology 62 (2012) 1708e1716
1711
3.6. Amantadine blocked NAT-mediated cytotoxicity more
potentlythan NMDA- and DAT-mediated cytoxicity
In order to investigate the long term activity of amantadine
onthe receptor or transporters cytotoxity assays were
established.
In cells expressing the human the NR1/2A under the control ofthe
T-REx� system, cytotoxicity was induced by treatment
withtetracycline for 24 h eliciting synthesis of the functional
NMDA-receptors which confer a detrimental effect presumably due
toconstant influx of calcium. Cytotoxic effects of drugs unrelated
tothe receptor were determined by exposing the cells to the drugs
inthe absence of tetracycline and were not observed for up to 0.3
mMof amantadine or ketamine and up to 0.1 mM of memantine(open
symbols in Fig. 7A). Ketamine and memantine blockedtetracycline
induced cytotoxicity concentration-dependently atconcentrations of
10 mM and above, whereas amantadine was onlyactive at concentration
of 0.3e1 mM (closed symbols in Fig. 7A).
Effects of drugs on NAT- or DAT-mediated cytotoxicity
weredetermined in cells expressing the human NAT or DAT under
thecontrol of the T-REx� system by exposing the cells to 1 mM
MPPþ
and tetracycline and measuring cell viability 72 h later
(closed
symbols in Fig. 7B,C); cytoxicity is due to the
transporter-mediatedaccumulation of the neurotoxin MPPþ. Potential
cytotoxic effects ofthe drugs themselves were measured in the
absence of MPPþ
(open symbols in Fig. 7B, C).In NAT expressing cells the NAT
inhibitor desipramine blocked
cytotoxicity in a concentration-dependent manner starting at0.1
mM, whereas the DAT inhibitor GBR 12909 was only weaklyprotective
at 3 mM. Amantadine blocked NAT-mediated MPPþ-cytoxicity
concentration-dependently starting with significanteffects at 30
mM. Viability of NAT expressing cells in the absence ofMPPþ was
decreased by 300 mM amantadine to 80% of control-treated cells
(open triangles in Fig. 7B) and viability in the pres-ence of MPPþ
was preserved by 300 mM amantadine to the samelevel (closed
triangles in Fig. 7B). Ketamine and memantine werenot protective in
concentrations up to 300 and 100 mM, respec-tively, concentrations
at which these drugs already displayeda weak toxic effect in the
absence of MPPþ.
In DAT expressing cells, the DAT inhibitor GBR 12909
blockedMPPþ-induced cytotoxicity at 0.03 mM and above,
restoringviability concentration-dependently up to control levels
(closedsymbols in Fig. 7C). Amantadine displayed considerable
cytotoxic
-
Fig. 3. Inhibition of uptake by amantadine in cells expressing
the human NAT or DAT.Concentration-inhibition curves for its effect
on [3H]-noradrenaline uptake in HEK293cells stably expressing the
human NAT (circles) or on [3H]-dopamine uptake in HEK293cells
stably expressing the human DAT (triangles). Specific activity of
the radiotracersamounted to about 0.375 Ci/mmol noradrenaline or
dopamine. Cells were incubated in24-well plates for 2.5 min at 37
�C with tritiated monoamines in the absence (control)or presence of
amantadine at the concentrations indicated, and uptake was
deter-mined as described under Methods. Symbols represent means �
SEM of 3e5 inde-pendent experiments, each in duplicates. The data
of each experiment were fitted bynonlinear regression, and the
means of the IC50 values � SEM are given in the Figure.
Fig. 4. Effect of amantadine in whole-cell patch-clamp
recordings of HEK293 cellsstably expressing the human NAT. Cells
were voltage-clamped at a holding potentialof �80 mV and superfused
for 2 (A, D) or 4 (B, C) seconds with the drugs(NA, noradrenaline)
indicated. In interaction experiments cocaine (A) or amantadine(D)
were already present in a pre-run of 2 s. Shown are mean values of
currenttraces � SEM (grey area) of 14e19 cells.
C. Sommerauer et al. / Neuropharmacology 62 (2012)
1708e17161712
effects in the absence of MPPþ at concentrations of 300 mM and1
mM, lowering viability to 76 and 56%, respectively (open symbolsin
Fig. 7C). In the presence of MPPþ, amantadine had a weak
butsignificant protective effect at 100 mM and restored viability
to 62%of control at 1 mM (closed symbols in Fig. 7C).
4. Discussion
In this study, we provide evidence that in vitro on intact,
livingcells amantadine has higher potency at the human NAT than at
thehuman NMDA-receptor: (1) in patch-clamp experiments 10
mMamantadine induced an inward current in NAT-expressing
cells,whereas 30 mM amantadine was inactive in blocking the
inwardcurrent induced by 100 mM NMDA in NMDA-receptor
expressingcells; (2) in cytotoxicity experiments � 30 mM amantadine
signifi-cantly blocked NAT-mediated cytotoxicity of MPPþ,
whereas300 mM amantadine were necessary to block
NMDA-receptormediated cytotoxicity.
We only examined cells expressing the human NR1/2A receptor,but
this receptor is abundant in the human striatum and highlyexpressed
in medium spiny neurons (Kosinski et al., 1998;Kuppenbender et al.,
2000), the main striatal projections neuronswithin the basal
ganglia circuit and driven by striatal NR2Asubunits under
parkinsonian conditions (Fantin et al., 2008). On theother hand,
only low levels of NR1/2A receptor were found inhuman globus
pallidus which receives in its internal segmenta glutamatergic
input from the subthalamic nucleus which isoveractive in
Parkinson’s disease and seems to be enriched inNR1/2D receptors
(Kosinski et al., 1998); still, differences in potencyof amantadine
reported at rat NR1A/2A and NR1A/2D receptorswere less than 3-fold
(Parsons et al., 1999), so at least equipotencyof amantadine at the
NAT and the NR1A/2D receptor can beexpected from our findings. The
potency of amantadine at thehuman NR1/2A on NMDA-induced currents
(estimatedIC50 ¼ 197 mM) was considerably lower than the values
reported forcultured cortical neurones from foetal rats or rat
NR1a/2A receptors
-
Fig. 5. Effect of amantadine in whole-cell patch-clamp
recordings of HEK293 cellsstably expressing the human DAT. Cells
were voltage-clamped at a holding potentialof �80 mV and superfused
for 2 (A, C) or 4 (B) seconds with the drugs(NA, noradrenaline; DA,
dopamine) indicated. In interaction experiments cocaine(A) or
amantadine (C) were already present in a pre-run of 2 s. Shown are
mean valuesof current traces � SEM (grey area) of 7e12 cells.
Fig. 6. Effect of amantadine on release by HEK293 cells stably
expressing the humanNAT. Cells grown on 5-mm-diameter coverslips
were preloaded with [3H]MPPþ andsuperfused at 25 �C with standard
Tris/HEPES buffer, and 4-min fractions werecollected. Bars indicate
fractions after exposure to 10 mM amphetamine or amantadineat the
concentrations indicated (A), 10 mM mazindol and 10 mM amphetamine
(B) or10 mMmazindol and 100 mM amantadine (C). Symbols represent
means � SEM of threeindependent experiments.
C. Sommerauer et al. / Neuropharmacology 62 (2012) 1708e1716
1713
expressed in Xenopus oocytes (IC50 ¼ 81 and 26 mM,
respectively;Parsons et al., 1999).
Amantadine blocked uptake by NAT expressing cells 30 foldmore
potently than uptake by DAT expressing cells. DAT blockadewas shown
to have antiparkinsonian effects in models of PD (Laneet al., 2005;
Madras et al., 2006). However, an IC50 of more than1 mM for the
blockade of DAT by amantadine in our experimentsmakes it rather
unlikely that interference with DAT function isrelevant for the
antiparkinsonian action of amantadine. In a studyon the organic
cation transporter OCT2 amantadine behaved as
substrate and competitive inhibitor for OCT2; the blockade of
OCT2was expected to result in increased extracellular dopamine
andtherefore was suggested as antiparkinsonian mechanism (Buschet
al., 1998). However, dopamine uptake and tissue content wasnot
different between OCT2�/� and wild-type mice in a recentreport
making the relevance of an interaction with the OCT2questionable
(Bacq et al., 2011).
Amantadine acted at the NAT in an amphetamine-like
mannerinducing reverse transport, however it was less effective
thanamphetamine. The translocation process at the DAT or NAT is
anelectrogenic process by cotransport of sodium ions resulting in
the
-
Fig. 7. Effect of amantadine on cytotoxic effects in HEK293
cells stably expressing thehuman NR1/2A receptor, NAT or DAT. Cells
expressing the NR1/2A receptor (A), the NAT(B) or DAT (C) under the
control of the T-REx� were seeded in 96-well plates, inducedby
treatment with tetracycline in the absence or presence of the drugs
at theconcentration indicated and under inclusion of 1 mM MPPþ in
NAT or DAT expressingcells (closed symbols). Effects of the drugs
unrelated to receptor or transporters (opensymbols) were determined
in the absence of tetracycline (A) or MPPþ (B, C). Cellviability
was determined by measuring acid phosphatase activity one (A) or
three (B, C)days later. Activity was expressed as percentage of
that of vehicle-treated cells.*p < 0.05, **p < 0.01, ***p
< 0.0001 vs zero drug by paired Student’s t-test followed
byBonferroni correction; symbols represent mean values � SEM of 4e9
independentexperiments, each in quadruplicates.
C. Sommerauer et al. / Neuropharmacology 62 (2012)
1708e17161714
influx of one or two positive charges for eachmolecule of
dopamineor noradrenaline pumped into the cell. Similar to the
transportersubstrate noradrenaline (Galli et al., 1995),
amphetamine inducesinward currents in NAT-expressing cells with
about the same
maximum effect in the low mmolar range as
noradrenaline(unpublished observation). In our study, the
amantadine-inducedinward-current plateaued at 10e100 mM, at a level
which wasabout 20% of the inward current induced by noradrenaline.
Inagreement with the hypothesis that substrate-induced
transportercurrents parallels substrate-induced
transporter-mediated release(Sitte et al., 1998),
amantadine-induced release also plateaued wellbelow the releasing
effect of amphetamine. It is interesting to notethat in superfusion
experiments there was no difference betweenthe releasing effect of
100 mM and 300 mM amantadine, whereas inthe toxicity assay on
NATexpressing cells 300 mM amantadine weremore effective than 100
mM. Two explanations are conceivable tojustify that high
concentrations are necessary for cytoprotection.(i) The mechanism
of cytoprotection is the blockade of the MPPþ
uptake into the cell. For induction of release however
amantadineacts as transporter substrate and elicits reverse
transport from thecell interior. We presume that with high
concentrations transport -both forward and reverse e may be
arrested which is in keepingwith the limited amount of MPPþ release
induced by amantadine(at lower concentrations). (ii) When NAT
expressing cells areco-incubated with amantadine and MPPþ, uptake
of amantadinewould be predictably outcompeted by MPPþ; MPPþ
affinity for theNAT was shown to exceed that of noradrenaline used
in the uptakeexperiment (Pifl et al., 1996).
An antiparkinsonian action of amphetamines has been reportedin
the literature (Miller and Nieburg, 1973; Parkes et al.,
1975;Solomon et al., 1937). Although amphetamines are
well-knownreleasers of dopamine, and dopamine release may be part
of itsantiparkinsonian action, amphetamines are even more
potentreleasers of noradrenaline (Rothman et al., 2001) and there
seemsto be a considerable contribution of noradrenaline to the
motoreffect of low dose amphetamine (Kuczenski and Segal, 2001;
Ögrenet al., 1983). Our experiments on NAT-expressing HEK293
cellssupport a releasing action directly via the NAT and rule out
anyindirect effects of amantadine via NMDA receptors as found
foreffects of amantadine on dopamine release in
microdialysisexperiments in the striatum (Quack et al., 1995;
Takahashi et al.,1996). Therapeutically active extracellular
concentrations ofamantadine were estimated to be in the low
micromolar range(Kornhuber et al., 1995); it appears reasonable to
speculate thoughthat an accumulation of amantadine in noradrenergic
nerveendings via the NAT - similarly to the accumulation of
amphet-amine in DAT-expressing HEK293 cells (Sitte et al., 1998) e
mayfavour an action on noradrenergic neurons. In a previous study
onthe noradrenaline and dopamine releasing action of amantadine
inrat tissue, 10 mM amanadine markedly increased isotope
outflowfrom superfused iris preincubated with tritiated
noradrenaline;effects on the on the dopamine-system were however
lessconvincing because they were inferred from a potentiating
effect ofamantadine on electrically-induced overflow of
radioactivity fromneostriatal slices preincubated with tritiated
dopamine (Farneboet al., 1971). Consistent with a noradrenergic
mechanism ofaction, amantadine (5 mg/kg i.p.) depleted
noradrenaline-containing vesicles in adrenal medullary cells of
rats andincreased noradrenaline plasma levels of humans 1e2 h after
oraladministration of 100 mg (Pita and Perez, 1977; Lechin et al.,
2010).
In principle, a higher potency of amantadine in
blockingNAT-mediated cytotoxicity of MPPþ than
NR1/2A-mediatedcytotoxicity may be due to distinct toxic mechanisms
of the twotoxic agents, namely MPPþ passing the NAT and cations
passing theNR1/2A receptor, mechanisms which could by distinctly
affected byamantadine. However, the much weaker potency of
amantadine inpreserving viability of MPPþ-exposed DAT cells
suggests that, infact, different affinities to transporters and
receptors are essentialfor these differences in potency.
-
C. Sommerauer et al. / Neuropharmacology 62 (2012) 1708e1716
1715
It is not clear which of amantadine’s pharmacological effects
areresponsible for its antiparkinsonian actions. Amantadine is the
onlydrug with relevant affinity to NMDA-receptors which is
establishedas a remedy against the motor symptoms of PD. Memantine
whichhas clearly higher affinity to NMDA-receptors than
amantadine(Parsons et al., 1995) does not appear to share the
antidyskineticactions of amantadine (Merello et al., 1999) and was
cited as anantiparkinsonian agent inferior to amantadine (Danysz et
al., 1997).Interestingly, in our study memantine did not
protectNAT-expressing cells from MPPþ-toxicity as observed for
amanta-dine which suggests differences between amantadine and
mem-antine on the noradrenergic system. The lower potency
ofamantadine in our cytotoxicity assays on NR1/2A
receptorexpressing cells as compared to patch-clamp recordings can
beexplained by the absence of Mg2þ in the
electrophysiologicalexperiments, whereas cytotoxicity assays were
performed in cellculture medium with Mg2þ-concentrations in the low
millimolarrange as to be expected in vivo. The relevance of
amantadine inhi-bition of NMDA receptors at clinical dosagewas
recently challengedby the Mg2þ-induced potency loss of amantadine
observed in two-electrode voltage-clamp recordings on oocytes
expressing humanNR1/2A receptors (Otton et al., 2011). NMDA
receptor inhibitionwasperceived not to be crucial in a recent study
on the neuroprotectionproduced by amantadine in culture models of
PD (Ossola et al.,2011), a study stimulated by indirect evidence of
neuroprotectionin a report on amantadine treatment as an
independent predictor ofimproved survival in PD (Uitti et al.,
1996). Our finding that NATrepresents a major molecular target
conforms to the proposedneuroprotective effect of amantadine;
pharmacological or geneticNAT blockade were reported to afford
protection of dopaminergicneurons in vivo (Rommelfanger and
Weinshenker, 2007).
In conclusion, although experiments on transfected cells
obvi-ously cannot establish amantadine’s mode of action in PD,
themolecular pharmacology as demonstrated in our study
appearscompatible with contributions from a noradrenergic
mechanism.
References
Adler, C.H., Stern, M.B., Vernon, G., Hurtig, H.I., 1997.
Amantadine in advancedParkinson’s disease: good use of an old drug.
J. Neurol 244, 336e337.
Bacq, A., Balasse, L., Biala, G., Guiard, B., Gardier, A.M.,
Schinkel, A., Louis, F.,Vialou, V., Martres, M.P., Chevarin, C.,
Hamon, M., Giros, B., Gautron, S., 2011.Organic cation transporter
2 controls brain norepinephrine and serotoninclearance and
antidepressant response. Mol. Psychiatry.
Bezard, E., Brefel, C., Tison, F., Peyro-Saint-Paul, H., Ladure,
P., Rascol, O., Gross, C.E.,1999. Effect of the alpha 2
adrenoreceptor antagonist, idazoxan, on motordisabilities in
MPTP-treated monkey. Prog. Neuropsychopharmacol. Biol.Psychiatry
23, 1237e1246.
Bofill-Cardona, E., Kudlacek, O., Yang, Q., Ahorn, H.,
Freissmuth, M., Nanoff, C., 2000.Binding of calmodulin to the
D2-dopamine receptor reduces receptor signalingby arresting the G
protein activation switch. J. Biol. Chem. 275, 32672e32680.
Busch, A.E., Karbach, U., Miska, D., Gorboulev, V., Akhoundova,
A., Volk, C., Arndt, P.,Ulzheimer, J.C., Sonders, M.S., Baumann,
C., Waldegger, S., Lang, F., Koepsell, H.,1998. Human neurons
express the polyspecific cation transporter hOCT2,
whichtranslocates monoamine neurotransmitters, amantadine, and
memantine. Mol.Pharmacol 54, 342e352.
Chen, H.S., Pellegrini, J.W., Aggarwal, S.K., Lei, S.Z., Warach,
S., Jensen, F.E.,Lipton, S.A., 1992. Open-channel block of
N-methyl-D-aspartate (NMDA)responses by memantine: therapeutic
advantage against NMDA receptor-mediated neurotoxicity. J. Neurosci
12, 4427e4436.
Connolly, D.T., Knight, M.B., Harakas, N.K., Wittwer, A.J.,
Feder, J., 1986. Determi-nation of the number of endothelial cells
in culture using an acid phosphataseassay. Anal. Biochem 152,
136e140.
Danysz, W., Parsons, C.G., Kornhuber, J., Schmidt, W.J., Quack,
G., 1997. Amino-adamantanes as NMDA receptor antagonists and
antiparkinsonian agentse-preclinical studies. Neurosci. Biobehav.
Rev 21, 455e468.
Dunn, J.P., Henkel, J.G., Gianutsos, G., 1986. Pharmacological
activity of amantadine:effect of N-alkyl substitution. J. Pharm.
Pharmacol 38, 353e356.
Fantin, M., Auberson, Y.P., Morari, M., 2008. Differential
effect of NR2A and NR2Bsubunit selective NMDA receptor antagonists
on striato-pallidal neurons:relationship to motor response in the
6-hydroxydopamine model of parkin-sonism. J. Neurochem 106,
957e968.
Farnebo, L.-O., Fuxe, K., Goldstein, M., Hamberger, B.,
Ungerstedt, U., 1971. Dopamineand noradrenaline releasing action of
amantadine in the central and peripheralnervous system: a possible
mode in Parkinson’s disease. Eur. J. Pharmacol. 16,27e38.
Fletcher, E.A., Redfern, P.H., 1970. The effect of amantadine on
the uptake ofdopamine and noradrenaline by rat brain homogenates.
J. Pharm. Pharmacol22, 957e959.
Galli, A., DeFelice, L.J., Duke, B.J., Moore, K.R., Blakely,
R.D., 1995. Sodium-dependentnorepinephrine-induced currents in
norepinephrine-transporter-transfectedHEK-293 cells blocked by
cocaine and antidepressants. J. Exp. Biol. 198,2197e2212.
Giros, B., el Mestikawy, S., Godinot, N., Zheng, K., Han, H.,
Yang-Feng, T., Caron, M.G.,1992. Cloning, pharmacological
characterization, and chromosome assignmentof the human dopamine
transporter. Mol. Pharmacol 42, 383e390.
Haacke, U., Sturm, G., Suwer, V., Wesemann, W., Wildenhahn, G.,
1977. [The action of1-aminoadamantane. Comparative studies with
isolated nerve endings andthrombocytes on the release of serotonin
and dopamine]. Arzneimittelfor-schung 27, 1481e1483.
Heikkila, R.E., Cohen, G., 1972. Evaluation of amantadine as a
releasing agent oruptake blocker for H 3 -dopamine in rat brain
slices. Eur. J. Pharmacol. 20,156e160.
Herblin, W.F., 1972. Amantadine and catecholamine uptake.
Biochem. Pharmacol 21,1993e1995.
Huber, T.J., Dietrich, D.E., Emrich, H.M., 1999. Possible use of
amantadine indepression. Pharmacopsychiatry 32, 47e55.
Jankovic, J., 2009. Atomoxetine for freezing of gait in
Parkinson disease. J. Neurol.Sci. 284, 177e178.
Kornhuber, J., Bormann, J., Hubers, M., Rusche, K., Riederer,
P., 1991. Effects of the1-amino-adamantanes at the MK-801-binding
site of the NMDA-receptor-gatedion channel: a human postmortem
brain study. Eur. J. Pharmacol 206, 297e300.
Kornhuber, J., Quack, G., Danysz, W., Jellinger, K., Danielczyk,
W., Gsell, W.,Riederer, P., 1995. Therapeutic brain concentration
of the NMDA receptorantagonist amantadine. Neuropharmacology 34,
713e721.
Kosinski, C.M., Standaert, D.G., Counihan, T.J., Scherzer, C.R.,
Kerner, J.A.,Daggett, L.P., Velicelebi, G., Penney, J.B., Young,
A.B., Landwehrmeyer, G.B., 1998.Expression of N-methyl-D-aspartate
receptor subunit mRNAs in the humanbrain: striatum and globus
pallidus. J.Comp Neurol. 390, 63e74.
Kuczenski, R., Segal, D.S., 2001. Locomotor effects of acute and
repeated thresholddoses of amphetamine and methylphenidate:
relative roles of dopamine andnorepinephrine. J. Pharmacol. Exp.
Ther 296, 876e883.
Kudlacek, O., Waldhoer, M., Kassack, M.U., Nickel, P., Salmi,
J.I., Freissmuth, M.,Nanoff, C., 2002. Biased inhibition by a
suramin analogue of A1-adenosinereceptor/G protein coupling in
fused receptor/G protein tandems: theA1-adenosine receptor is
predominantly coupled to Goalpha in human brain.Naunyn
Schmiedebergs Arch. Pharmacol. 365, 8e16.
Kuppenbender, K.D., Standaert, D.G., Feuerstein, T.J., Penney
Jr., J.B., Young, A.B.,Landwehrmeyer, G.B., 2000. Expression of
NMDA receptor subunit mRNAs inneurochemically identified projection
and interneurons in the human striatum.J. Comp Neurol. 419,
407e421.
Laitinen, L., 1969. Desipramine in treatment of Parkinson’s
disease. A placebo-controlled study. Acta Neurol. Scand 45,
109e113.
Lane, E.L., Cheetham, S., Jenner, P., 2005. Dopamine uptake
inhibitor-inducedrotation in 6-hydroxydopamine-lesioned rats
involves both D1 and D2 recep-tors but is modulated through
5-hydroxytryptamine and noradrenalinereceptors. J. Pharmacol. Exp.
Ther 312, 1124e1131.
Lechin, F., van der Dijs, B., Parde-Maldonado, B., Rivera, J.E.,
Baez, S., LechinM., E.,2010. Effects of amantadine on circulating
neurotransmitters in healthysubjects. J. Neural. Transm 117,
293e299.
Lupp, A., Lucking, C.H., Koch, R., Jackisch, R., Feuerstein,
T.J., 1992. Inhibitory effectsof the antiparkinsonian drugs
memantine and amantadine on N-methyl-D-aspartate-evoked
acetylcholine release in the rabbit caudate nucleus in vitro.J.
Pharmacol. Exp. Ther 263, 717e724.
Lustig, H.S., Ahern, K.V., Greenberg, D.A., 1992.
Antiparkinsonian drugs and in vitroexcitotoxicity. Brain Res. 597,
148e150.
Madras, B.K., Fahey, M.A., Goulet, M., Lin, Z., Bendor, J.,
Goodrich, C., Meltzer, P.C.,Elmaleh, D.R., Livni, E., Bonab, A.A.,
Fischman, A.J., 2006. Dopamine transporter(DAT) inhibitors
alleviate specific parkinsonian deficits in monkeys:
associationwith DAT occupancy in vivo. J. Pharmacol. Exp. Ther 319,
570e585.
Marsh, L., Biglan, K., Gerstenhaber, M., Williams, J.R., 2009.
Atomoxetine for thetreatment of executive dysfunction in
Parkinson’s disease: a pilot open-labelstudy. Mov. Disord 24,
277e282.
Merello, M., Nouzeilles, M.I., Cammarota, A., Leiguarda, R.,
1999. Effect ofmemantine (NMDA antagonist) on Parkinson’s disease:
a double-blind cross-over randomized study. Clin. Neuropharmacol
22, 273e276.
Miller, E., Nieburg, H.A., 1973. Amphetamines, valuable adjunct
in treatment ofParkinsonism. N.Y. State J. Med 73, 2657e2661.
Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M.,
Lomeli, H.,Burnashev, N., Sakmann, B., Seeburg, P.H., 1992.
Heteromeric NMDA receptors:molecular and functional distinction of
subtypes. Science 256, 1217e1221.
Narabayashi, H., Kondo, T., Yokochi, F., Nagatsu, T., 1987.
Clinical effects of L-threo-3,4-dihydroxyphenylserine in cases of
parkinsonism and pure akinesia.Adv. Neurol 45, 593e602.
Ögren, S.O., Archer, T., Johansson, C., 1983. Evidence for a
selective brain norad-renergic involvement in the locomotor
stimulant effects of amphetamine in therat. Neurosci. Lett. 43,
327e331.
-
C. Sommerauer et al. / Neuropharmacology 62 (2012)
1708e17161716
Ossola, B., Schendzielorz, N., Chen, S.H., Bird, G.S., Tuominen,
R.K., Mannisto, P.T.,Hong, J.S., 2011. Amantadine protects dopamine
neurons by a dual action:reducing activation of microglia and
inducing expression of GNDF in astroglia.Neuropharmacology 61,
574e582.
Otton, H.J., Lawson, M.A., Pannozzo, M.A., Davies, C.H., Wyllie,
D.J., 2011. Quantifi-cation of the Mg(2þ)-induced potency shift of
amantadine and memantinevoltage-dependent block in human
recombinant GluN1/GluN2A NMDARs.Neuropharmacology 60, 388e396.
Paczkowski, F.A., Bryan-Lluka, L.J., Porzgen, P., Bruss, M.,
Bonisch, H., 1999.Comparison of the pharmacological properties of
cloned rat, human, andbovine norepinephrine transporters. J.
Pharmacol. Exp. Ther 290, 761e767.
Papeschi, R., 1974. Amantadine may stimulate dopamine and
noradrenalinereceptors. Neuropharmacology 13, 77e83.
Parkes, J.D., Calver, D.M., Zilkha, K.J., Knill-Jones, R.P.,
1970. Controlled trial ofamantadine hydrochloride in Parkinson’s
disease. Lancet 1, 259e262.
Parkes, J.D., Tarsy, D., Marsden, C.D., Bovill, K.T., Phipps,
J.A., Rose, P., Asselman, P.,1975. Amphetamines in the treatment of
Parkinson’s disease. J. Neurol.Neurosurg. Psychiatry 38,
232e237.
Parsons, C.G., Danysz, W., Bartmann, A., Spielmanns, P.,
Frankiewicz, T.,Hesselink, M., Eilbacher, B., Quack, G., 1999.
Amino-alkyl-cyclohexanes arenovel uncompetitive NMDA receptor
antagonists with strong voltage-dependency and fast blocking
kinetics: in vitro and in vivo characterization.Neuropharmacology
38, 85e108.
Parsons, C.G., Panchenko, V.A., Pinchenko, V.O., Tsyndrenko,
A.Y., Krishtal, O.A.,1996. Comparative patch-clamp studies with
freshly dissociated rat hippo-campal and striatal neurons on the
NMDA receptor antagonistic effects ofamantadine and memantine. Eur.
J. Neurosci 8, 446e454.
Parsons, C.G., Quack, G., Bresink, I., Baran, L., Przegalinski,
E., Kostowski, W.,Krzascik, P., Hartmann, S., Danysz, W., 1995.
Comparison of the potency, kineticsand voltage-dependency of a
series of uncompetitive NMDA receptor antago-nists in vitro with
anticonvulsive and motor impairment activity in
vivo.Neuropharmacology 34, 1239e1258.
Pifl, C., Hornykiewicz, O., Giros, B., Caron, M.G., 1996.
Catecholamine transportersand
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity:
studiescomparing the cloned human noradrenaline and human dopamine
transporter.J. Pharmacol. Exp. Ther 277, 1437e1443.
Pifl, C., Khorchide, M., Kattinger, A., Reither, H., Hardy, J.,
Hornykiewicz, O., 2004a.Alpha-Synuclein selectively increases
manganese-induced viability loss in SK-N-MC neuroblastoma cells
expressing the human dopamine transporter.Neurosci. Lett. 354,
34e37.
Pifl, C., Rebernik, P., Kattinger, A., Reither, H., 2004b. Zn2þ
modulates currentsgenerated by the dopamine transporter: parallel
effects on amphetamine-induced charge transfer and release.
Neuropharmacology 46, 223e231.
Pita, E., Perez, N., 1977. Ultrastructural evidence of
amantadine and amphetaminenoradrenaline releasing action.
Experientia 33, 72e74.
Quack, G., Hesselink, M., Danysz, W., Spanagel, R., 1995.
Microdialysis studies withamantadine and memantine on
pharmacokinetics and effects on dopamineturnover. J. Neural Transm.
Suppl. 46, 97e105.
Rajput, A.H., Rajput, A., Lang, A.E., Kumar, R., Uitti, R.J.,
Galvez-Jimenez, N., 1998.New use for an old drug: amantadine
benefits levodopa-induced dyskinesia.Mov Disord. 13, 851.
Rascol, O., Arnulf, I., Peyro-Saint, P.H., Brefel-Courbon, C.,
Vidailhet, M., Thalamas, C.,Bonnet, A.M., Descombes, S., Bejjani,
B., Fabre, N., Montastruc, J.L., Agid, Y., 2001.Idazoxan, an
alpha-2 antagonist, and L-DOPA-induced dyskinesias in patientswith
Parkinson’s disease. Mov Disord. 16, 708e713.
Rommelfanger, K.S., Weinshenker, D., 2007. Norepinephrine: the
redheaded step-child of Parkinson’s disease. Biochem. Pharmacol.
74, 177e190.
Rothman, R.B., Baumann, M.H., Dersch, C.M., Romero, D.V., Rice,
K.C., Carroll, F.I.,Partilla, J.S., 2001. Amphetamine-type central
nervous system stimulantsrelease norepinephrine more potently than
they release dopamine and sero-tonin. Synapse 39, 32e41.
Ruzicka, E., Streitova, H., Jech, R., Kanovsky, P., Roth, J.,
Rektorova, I., Mecir, P.,Hortova, H., Bares, M., Hejdukova, B.,
Rektor, I., 2000. Amantadine infusion intreatment of motor
fluctuations and dyskinesias in Parkinson’s disease. J.
NeuralTransm. 107, 1297e1306.
Savola, J.M., Hill, M., Engstrom, M., Merivuori, H., Wurster,
S., McGuire, S.G., Fox, S.H.,Crossman, A.R., Brotchie, J.M., 2003.
Fipamezole (JP-1730) is a potent alpha2adrenergic receptor
antagonist that reduces levodopa-induced dyskinesia in
theMPTP-lesioned primate model of Parkinson’s disease. Mov Disord.
18, 872e883.
Sawada, H., Oeda, T., Kuno, S., Nomoto, M., Yamamoto, K.,
Yamamoto, M.,Hisanaga, K., Kawamura, T., 2010. Amantadine for
dyskinesias in Parkinson’sdisease: a randomized controlled trial.
PLoS One 5, e15298.
Scatton, B., Cheramy, A., Besson, M.J., Glowinski, J., 1970.
Increased synthesis andrelease of dopamine in the striatum of the
rat after amantadine treatment. Eur.J. Pharmacol. 13, 131e133.
Schwab, R.S., England Jr., A.C., Poskanzer, D.C., Young, R.R.,
1969. Amantadine in thetreatment of Parkinson’s disease. JAMA 208,
1168e1170.
Sitte, H.H., Huck, S., Reither, H., Boehm, S., Singer, E.A.,
Pifl, C., 1998. Carrier-medi-ated release, transport rates, and
charge transfer induced by amphetamine,tyramine, and dopamine in
mammalian cells transfected with the humandopamine transporter. J.
Neurochem 71, 1289e1297.
Solomon, P., Mitchell, R.S., Prinzmetal, M., 1937. The use of
benzedrie sulfate inpostencephalitic Parkinson’s disease. J. Am.
Med. Assoc. 108, 1765e1770.
Takahashi, T., Yamashita, H., Zhang, Y.X., Nakamura, S., 1996.
Inhibitory effect ofMK-801 on amantadine-induced dopamine release
in the rat striatum. BrainRes. Bull. 41, 363e367.
Thomas, A., Iacono, D., Luciano, A.L., Armellino, K., Di, I.A.,
Onofrj, M., 2004. Durationof amantadine benefit on dyskinesia of
severe Parkinson’s disease. J. Neurol.Neurosurg. Psychiatry 75,
141e143.
Thornburg, J.E., Moore, K.E., 1973. Dopamine and norepinephrine
uptake by ratbrain synaptosomes: relative inhibitory potencies of
1- and d-amphetamineand amantadine. Res. Commun. Chem. Pathol.
Pharmacol. 5, 81e89.
Tohgi, H., Abe, T., Takahashi, S., 1993. The effects of
L-threo-3,4-dihydrox-yphenylserine on the total norepinephrine and
dopamine concentrations in thecerebrospinal fluid and freezing gait
in parkinsonian patients. J. Neural Transm.Park Dis. Dement. Sect
5, 27e34.
Uitti, R.J., Rajput, A.H., Ahlskog, J.E., Offord, K.P.,
Schroeder, D.R., Ho, M.M.,Prasad, M., Rajput, A., Basran, P., 1996.
Amantadine treatment is an independentpredictor of improved
survival in Parkinson’s disease. Neurology 46, 1551e1556.
Verhagen, M.L., Del, D.P., van den, M.P., Fang, J., Mouradia,
M.M., Chase, T.N., 1998.Amantadine as treatment for dyskinesias and
motor fluctuations in Parkinson’sdisease. Neurology 50,
1323e1326.
The noradrenaline transporter as site of action for the
anti-Parkinson drug amantadine1. Introduction2. Methods2.1. Cell
culture and molecular biology2.2. Determination of cAMP
formation2.3. Patch clamp experiments2.4. Uptake of monoamines2.5.
Assay of reverse transport2.6. Cell viability
3. Results3.1. Amantadine had no affinity for α2
adrenoceptors3.2. Amantadine blocked NMDA-induced currents at
concentrations beyond 30 μM3.3. Amantadine blocked the NAT more
potently than the DAT3.4. Amantadine induced inward currents in NAT
expressing cells3.5. Amantadine induced NAT-mediated release3.6.
Amantadine blocked NAT-mediated cytotoxicity more potently than
NMDA- and DAT-mediated cytoxicity
4. DiscussionReferences